Journal of the American Association for Laboratory Animal Science Vol 55, No 2

Copyright 2016 March 2016

by the American Association for Laboratory Animal Science Pages 221–223

Measurement of Tear Production in English

Angora and Dutch Rabbits
Seyed Mehdi Rajaei,1 Siamak Mashhady Rafiee,1,* Masoud Selk Ghaffari2, Mohammad N Masouleh,1 and Mahmoud
Jamshidian3

The purpose of this study was to establish normal values for tear production tests in different breeds of domestic rabbits.
Healthy adult rabbits (n = 60; 120 eyes) of 2 different breeds (English angora and Dutch; n = 15 of each sex and breed) were
used in this study. Tear production was measured by using the 1-min Schirmer tear test (STT), phenol red thread test (PRTT),
and endodontic absorbent paper point tear test (EAPTT). In addition, horizontal palpebral fissure length was evaluated as a
measure of ocular adnexal dimensions. Tear production (mean ± 1 SD) in English angora rabbits was 5.4 ± 1.6 mm/min accord-
ing to the STT, 25.0 ± 2.7 mm in 15 s for the PRTT, and 18.8 ± 2.1 mm/min by the EAPTT; in Dutch rabbits, these values were
4.6 ± 1.2 mm/min, 23.6 ± 2.3 mm in 15 s, and 16.9 ± 1.7 mm/min, respectively. Only the EAPTT revealed a significant difference
in tear production between English Angora and Dutch rabbits. These results provide reference values for tear production in
English Angora and Dutch rabbits according to 3 different quantitative tear film assessment methods.

Abbreviations: EAPTT, endodontic absorbent paper tear test; HPFL, horizontal palpebral fissure length; PRTT, phenol red thread
test; STT, Schirmer tear test

Domestic rabbits (Oryctolagus cuniculus) are popular pets and
commonly referred to veterinarians for routine health care and
solving health problems. Rabbits are also common laboratory
research animals, because they are tractable and inexpensive
to maintain. Ocular lesions of all segments of the eyes have
been reported in wild, pet, and research animals.2 Rabbits have
prominent, laterally placed eyes, allowing them a large field
of vision, which is typical of prey species. They have an upper
eyelid, a larger and thinner lower eyelid, and a third eyelid (or
nictitating membrane). Rabbits normally blink infrequently,
averaging 10 to 12 times hourly.12
The tear film is as vital to the normal function of the eyes as
any anatomic component and is essential in the maintenance
of corneal clarity.10,13 The aqueous layer is responsible for
lubricating, oxygenating, and nourishing the cornea; flushing
debris from the corneal surface; providing various antibacterial
properties. The mucin layer allows the aqueous tear fluid to
spread evenly over the cornea and allows the adherence of the
tear film to the corneal surface.5 The lipid layer of tears contains
waxy esters, sterols, triglycerides, cholesterol, and some polar
lipids. This lipid mixture coats the surface of the tear film and
retards tear evaporation.13
Quantitative tests for the evaluation of tear film include
the Schirmer tear test (STT), phenol red tear test (PRTT), and
endodontic absorbent paper point tear test (EAPTT). The STT
is the most commonly used, standard method for tear film as-
sessment. The results of various ophthalmic diagnostic tests
have been reported for New Zealand white rabbits1,3,11 but
not for any other breed. The purpose of the current study was
Received: 11 May 2015. Revision requested: 12 Jun 2015. Accepted: 17 Jun 2015.
1Department of Clinical Sciences. Faculty of Specialized Veterinary Sciences, Science and
Research Branch, Islamic Azad University, Tehran, Iran, and 2Department of Clinical
Sciences, College of Veterinary Medicine, Karaj Branch, Alborz, Iran, and 3Department of
Basic Sciences, Faculty of Specialized Veterinary Sciences, Science and Research Branch,
Islamic Azad University, Tehran, Iran.
*Corresponding author. Email: Sr1vet@yahoo.com
to establish normal values for tear production in 2 breeds of
domestic rabbits, English angora and Dutch, according to the
STT, PRTT, and EAPTT.
Materials and Methods
Animals. The study was approved by Iran Society for Pre-
vention of Cruelty to Animals in accordance with Iranian
ethical codes for studies on laboratory animals. The popula-
tion consisted of 60 adult healthy rabbits (120 eyes) ranging
in age from 1 to 2.5 y of 2 different breeds, English angora
and Dutch; 15 male and 15 female rabbits from each breed
were examined. All of the rabbits weighed between 1470
and 3680 g (mean ± 1 SD, 2514 ± 482 g) and were housed
individually indoors beginning 7 d before the first test day
in an air conditioned room at constant temperature (18 to 21
°C) and humidity (45% to 48%). The rabbits were exposed to
a 12:12-h light:dark cycle, were fed a commercial rabbit diet,
and had unrestricted access to water.
Study procedures. The studied population was selected
on the basis of normal physical and ophthalmic examina-
tion including CBC analysis, radiography, ophthalmoscopy
(Binocular Indirect Ophthalmoscope, Welch Allyn, Rochester,
NY), fluorescein staining (Fluorescein Glostrips, Nomax, St
Louis, MO), slit lamp biomicroscopy (PSL Portable Slit Lamp,
Reichert, Buffalo, NY), and tonometry (TonoVet, iCare, Tiolat,
Helsinki, Finland).
Phenol red test threads (Zone-Quick, Menicon America, San
Mateo, CA), endodontic absorbent paper points (Roeko Color,
Roeko, Langenau, Germany), and Schirmer tear test strips
(Opstrip, Ophtechnics, Haryana, India) from a single lot were
used. A single examiner (SMR) performed all the ocular tests,
examinations, and measurements. All tests were performed
between 1400 and 1900 to minimize any possible variations
associated with diurnal changes.
On day 1, PRTT was performed. The lower eyelid of each
rabbit was everted, and the 3-mm folded head of the PRTT

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Vol 55, No 2
Journal of the American Association for Laboratory Animal Science
March 2016
cotton thread was placed into the ventral conjunctival fornix
for 15 s. The thread was then removed, and the portion of
the thread that had become red in color was measured (in
millimeters). On day 3, aqueous tear volume was measured
using EAPPTT. One absorbent paper point was inserted in the
lower conjunctival fornix of each eye; after 1 min, the paper
point was removed, and its wetted portion was immediately
measured by using a digital caliper graduated in millimeters.
On day 5, an STT strip was placed in the inferior conjunctival
fornix of one eye; after 30 s, another strip was placed in the
contralateral eye. After 1 min, the strips were removed, and
the amount of wetting was recorded (in millimeters per min-
ute). In addition, the distance between the inner end of the
caruncle and the inner end of the temporal canthus (that is,
the horizontal palpebral fissure length [HPFL]) was measured
by using a digital caliper.
Statistical analysis. Statistical analysis was performed by
using SPSS for Microsoft Windows (SPSS 20.0, IBM, Chicago,
IL). Normality was tested by a one-sample Kolmogorov–
Smirnov test. A paired-samples t test was used to compare
PRTT, EAPTT, STT, and HPFL values obtained from the right
and left eyes. Means and SD were calculated for left and right
eyes individually and for all eyes combined. An independent-
samples t test was used to compare mean PRTT, EAPTT, STT,
and HPFL values according to sex and body weight in each
group. Spearman correlation was used to evaluate the rela-
tionship between mean PRTT, EAPTT, STT, and HPFL values
and body weight. A P value less than 0.05 was considered
statistically significant.
Results
No rabbit showed any sign of ocular discomfort during the
24 h immediately after each tear production test. In addition,
all rabbits were examined on day 17, and all were free of signs
of conjunctivitis, keratitis, blepharitis, corneal ulceration, and
intraocular diseases.
All of the continuous numeric data obtained from the
population used in this investigation were normally distrib-
uted according to the one-sample Kolmogorov–Smirnov test
(P > 0.2). Body weights (mean ± 1 SD) of English angora and
Dutch rabbits were 2775 ± 461 g and 2254 ± 357 g (P = 0.01),
respectively. For English angora rabbits, tear production was
5.4 ± 1.6 mm/min according to the STT, 25.0 ± 2.7 mm in 15 s
by the PRTT, and 18.8 ± 2.1 mm/min in the EAPTT; for Dutch
rabbits, these values were 4.6 ± 1.2 mm/min, 23.6 ± 2.3 mm
in 15 s, and 16.9 ± 1.7 mm/min, respectively (Figure 1 and
Table 1). For all tests, tear production did not differ between
the left and right eyes or between male and female rabbits.
Tear production according to EAPTT and HPFL differed
significantly between English Angora and Dutch rabbits (P
= 0.043 and 0.007, respectively); HPFL values were 16.0 ± 0.7
mm for English angora rabbits and 12.9 ± 2.8 mm for Dutch
rabbits. There was no correlation between tear production
values and the body weight or age of the rabbits. However
the age and body weight were positively correlated (Pearson
r = 0.708, P = 0.001; Spearman r = 0.638, P = 0.003), and both
body weight (r = 0.637, P = 0.003) and HPFL (r = 0.669, P =
0.001) were positively correlated with the age of the rabbits.
In addition, tear production values from the PRTT and STT
were correlated (Pearson r = 0.543, P = 0.013; Spearman r =
0.511, P = 0.021) but not those from the PRTT and EAPTT or
EAPTT and STT (Table 2).
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Figure 1. Box-and-whisker plots of tear production in the study pop-
ulation of 60 English angora and Dutch rabbits. STT, Schirmer tear
test(mm/min); PRTT, phenol red thread test(mm/15s); EAPTT, endo-
dontic absorbent paper tear test(mm/min).
Discussion
Rabbits have a large bilobed lacrimal gland (located
ventrally behind the lower eyelid and adjacent to the lower
orbital rim), Harderian gland (attached to the lower medial
orbital wall), gland of the third eyelid, and venous sinus
within each orbit.8 The lacrimal gland, Harderian gland, and
gland of the third eyelid supply components of the precorneal
tear film that are crucial to the health of the cornea.12 Rab-
bits are somewhat unique in having a single nasolacrimal
punctum deep in the ventromedial conjunctival fornix. The
nasolacrimal duct follows a tortuous route with several
sudden constrictions, which are often sites of obstruction.4
Interbreed differences in the normal ophthalmic anatomy of
rabbits have not yet been described. In the current study, all
of the quantitative tear film assessment tests showed greater
tear production in English angora rabbits than in Dutch
rabbits. In addition, the HPFL was larger in English angora
than Dutch rabbits. Although HPFL and blink frequency
might directly affect the measurement of tear production
and spread of tear film,9 whether this is the case has yet to be
evaluated. In addition, fitting a conventional STT strip into a
small palpebral fissure might be difficult and thus affect the
measurement of tear production by this method. However,
the rabbits in the current study had a mean HPFL of 12.98
mm, and it was easy to use standard STT strips.
For New Zealand white rabbits, normal tear production has
been reported as 4.8 ± 2.9 to 5.3 ± 2.9 by STT,1,3,11 20.8 ± 3.7 by
PRTT,10 and 13.8 ± 1.5 mm/min by EAPTT,11 respectively. For
the STT, the tear production values of English angora and Dutch
rabbits were close to the reference range for New Zealand White
rabbits. However, tear production according to the PRTT and
EAPTT was greater in both of the breed we tested than that
reported for New Zealand White rabbits.
Although nasolacrimal outflow during STT measurement
is reduced to zero, this situation does not compromise the
determination of tear production. The STT strip absorbs all
of the tears produced, and tear uptake by the test strip equals
the tear production by the lacrimal and Harderian glands and
gland of the third eyelid.14 In contrast, the argument might
be made that PRTT and EAPTT measure tear volume in the
conjunctival sac rather than assessing de novo tear production
by lacrimal glands.
The parasympathetic nervous system provides the primary
control of aqueous tear secretion. Basal secretion of tears is pre-
sent continuously, and the increases in tear flow are stimulated
by the nervous system.6 Although tear production is affected
by environmental factors, time of measurement, and age of
animals,7 we controlled for these factors in the current study.
Future studies on the normal ocular anatomy of different breeds
 Tear production in rabbits

Table 1. Tear production in 60 rabbits, measured bilaterally

STT (mm/min) PRTT (mm in 15 s) EAPTT (mm/min)
Rabbits Mean ± 1 SD Range Mean ± 1 SD Range Mean ± 1 SD Range
Male 5.5 ± 1.3 3.5–7.5 25.3 ± 1.8 22.5–29.0 17.3 ± 2.4 14.5–22.0
Female 4.3 ± 1.5 2.5–7.0 23.0 ± 2.9 17.5–26.5 18.4 ± 1.5 15.0–21.0

English angora 5.4 ± 1.6 2.5–7.5 25.0 ± 2.7 24.0–29.0 18.8 ± 2.1a 16.5–22.0
Dutch 4.6 ± 1.2 3.0–6.5 23.6 ± 2.3 17.5–26.0 16.9 ± 1.7a 14.5–19.0
STT, Schirmer tear test; PRTT, Phenol red thread test; EAPTT, endodontic absorbent paper tear test
aThese values are significantly different (P = 0.043).

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